European Journal of Pharmacology 609 (2009) 126–131 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar Pulmonary, Gastrointestinal and Urogenital Pharmacology Effects of umbelliferone in a murine model of allergic airway inflammation Juliana F. Vasconcelos a,e, Mauro M. Teixeira b, José M. Barbosa-Filho c, Maria F. Agra c, Xirley P. Nunes d, Ana Maria Giulietti e, Ricardo Ribeiro-dos-Santos a,f, Milena B.P. Soares a,f,⁎ a Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, 40296-750, Salvador, BA, Brazil b Instituto de Ciências Biomédicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil c Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, 58051-970, João Pessoa, PB, Brazil d Colegiado do Curso de Medicina, Universidade Federal do Vale do São Francisco, 56306-410, Petrolina, PE, Brazil e Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, 44031-640, Feira de Santana, BA, Brazil f Hospital São Rafael. Av. São Rafael, 2152. São Marcos 41253-190, Salvador, BA, Brazil article info abstract Article history: The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis Received 7 December 2008 (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and Received in revised form 19 February 2009 challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a Accepted 3 March 2009 marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In Available online 14 March 2009 addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-γ, was found in bronchoalveolar lavage fluids of mice treated with Keywords: fi Umbelliferone umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-speci cIgEwerenot Coumarin significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone Typha domingensis attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of Bronquic asthma action of this molecule may contribute for the development of new drugs for the treatment of asthma. (Mouse) © 2009 Elsevier B.V. All rights reserved. 1. Introduction Apiaceae (Umbelliferae) family such as carrot, coriander and garden angelica, as well as plants from other families such as the mouse-ear Coumarins constitute a very large class of compounds present in hawkweed. It is a yellowish-white crystalline solid which has a slight several species belonging to different botanical families, which are solubility in hot water, but high solubility in ethanol (Dean, 1963). widespread in the world. They are secondary metabolites naturally Asthma is a chronic inflammatory disorder of the airways in which occurring in different parts of the plants, such as roots, flowers and fruits many cells and elements play a role, associated with increased airway (Leal et al., 2000; Ribeiro and Kaplan, 2002; Razavi et al., 2008). The search hyperresponsiveness, leading to recurrent episodes of wheezing, breath- for useful pharmaceutical has led to a resurgence of interest in coumarins lessness, chest tightness, and coughing (Barnes, 2001). This chronic because these substances display potent and relevant pharmacological inflammation is characterized by infiltration of eosinophils, neutrophils, activities that are structure-dependent, while at the same time appearing mast cells and T cells (Casale et al., 1987; Wenzel, 2003), and is promoted to lack toxicity in mammalian systems (Hoult and Payá, 1996). by Th2 type immune responses (Barnes, 2001; El Biaze et al., 2003; Reed, Among the biological effects of coumarins are anti-microbian (Thati 2006). In the current study we aimed to evaluate the effects of et al., 2007), anti-viral (Mazzei et al., 2008), anti-thrombotic, vasodilatory umbelliferone, a coumarin purified from Typha domingensis (Typhaceae), (Casley-Smith et al., 1993), antitumoral, antineoplasic, antiinflammatory in a mouse model of asthma, as a potential anti-asthmatic agent. (Hoult and Payá, 1996; Lino et al., 1997), and antimetastatic properties (Jiménez-Orozco et al., 2001; Finn et al., 2004; Finn et al., 2005; Elinos- Báez et al., 2005), and inhibition of acetylcholinesterase and angiotensin 2. Materials and methods converting enzyme (Barbosa-Filho et al., 2006a,b). In humans, coumarins have a short half-life, and its major biotransformation product (75%) is 2.1. Plant material umbelliferone (Hoult and Payá, 1996; Egan et al., 1997). Umbelliferone or 7-hydroxycoumarin is a widespread natural product The aerial parts of T. domingensis Pers. were collected for first of the coumarin family (Fig. 1). It occurs in many familiar plants from the extracts in March, 2002 in Bravo, State of Bahia, in temporary shallow lake in Dryland “Caatinga” Bioma and after in December, 2005 near the city of Santa Rita, State of Paraíba, Brazil. The plant was identified ⁎ Corresponding author. Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, by Ana Maria Giulietti and Maria de Fátima Agra and the voucher Laboratório de Engenharia Tecidual e Imunofarmacologia. Rua Waldemar Falcão, 121, Candeal. 40296-710, Salvador, Bahia, Brazil. Tel.: +55 71 3176 2260; fax: +55 71 31762272. specimens (Giulietti 2035 et al. and Agra 5520 et Góis — JPB) were E-mail address: milena@bahia.fiocruz.br (M.B.P. Soares). deposited in the Herbaria HUEFS and JPB. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.03.027 J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131 127 2.4. Sensitization and challenge with ovalbumin and treatment Allergic airway inflammation was induced as described before (Vasconcelos et al., 2008). Groups of seven mice received systemic immunization by subcutaneous injection of 10 μg of chicken egg ovalbumin (Grade V, N98% pure; Sigma, St Louis, MO) diluted in Fig. 1. Chemical structure of umbelliferone. 2 mg/ml alum (AlumImject; Pierce, Rockford, IL) followed by a booster injection at day 14. A nasal challenge was performed starting at day 28, by inhalational exposure to aerosolised ovalbumin for 15 min/day, on five consecutive days. Exposures were carried out in an acrylic box. A 2.2. Extraction and isolation of umbelliferone solution of 1% ovalbumin in saline was aerosolised by delivery of compressed air to a sidestream jet nebuliser (RespiraMax, NS, Brazil). The aerial parts (2000 g) of T. domingensis were extracted with 95% Two hours before each aerosol delivery, mice were treated orally with ethanol at room temperature. The extract (150 g) was evaporated under umbelliferone (30, 60, and 90 mg/kg), dexamethasone (30 mg/kg) or vacuum to yield a brown residue which was suspended in methanol: vehicle (10% DMSO in saline). H2O (3:7 v/v) and fractionated with chloroform and ethyl acetate. The chloroform phase (25 g) was subjected to column chromatography over 2.5. Collection of blood and bronchoalveolar lavage silica gel, eluted with hexane, chloroform and methanol mixtures in an increasing order of polarity, yielding three coumarins and one cinnamic Twenty four hours after the last inhalational exposure, mice were acid derivative. The ethyl acetate phase (25 g) was subjected to anesthetized and bled via the brachial plexus for collection of blood chromatography column over Sephadex LH-20 using methanol yielding samples used to estimate the IgE production. Bronchoalveolar lavage fl fi two avonoids. The isolated compounds were identi ed as coumarin (BAL) was performed twice by intratracheal instillation of 1 ml of PBS. (0.0044%), scopoletin (0.0014%) (Brateoeff and Perez-Amador, 1994), The first lavage fluid was centrifuged, and aliquots of the supernatant umbelliferone (0.007%) (Cussans and Huckerby, 1975), p-coumaric acid were kept at 70 °C until use for cytokine measurements. The second (0.0016%), (Pouchert, 1992), quercetin (0.0028%) (Williams et al., 1971) lavage fluid was centrifuged and the two cell pellets were resus- and isorhamnetin-3-O-glucoside (0.0039%) (Bilia et al., 1994) based on pended in a PBS final volume of 1 ml. The number of total leukocytes 1 13 HNMR, C NMR, COSY, HMQC and HMBC spectroscopic data and in bronchoalveolar lavage fluid was estimated in a Neubauer chamber. comparison with those reported in the literature. Differential counts were obtained using panotic-stained cytospin preparations. A differential count of 200 cells was made in a blinded 2.3. Animals fashion and according to standard morphologic criteria. Male BALB/c mice, 4–6 weeks old, were used in the experiments. 2.6. Histopathological and morphometric analysis All animals were raised and maintained at the animal facilities of the Gonçalo Moniz Research Center, FIOCRUZ/BA, in rooms with con- The right lobe of the lungs from each animal was removed for trolled temperature (22±2 °C) and humidity (55±10%) and histological analysis. The lung was inflated via the tracheal cannula continuous air renovation. Animals were housed in a 12 h light/12 h with 4% buffered formalin, fixed in the same solution, and embedded dark cycle (6 am–6 pm) and provided with rodent diet and water ad in paraffin. Sections were stained with hematoxylin and eosin for libitum. Animals were handled according to the NIH guidelines for quantification of inflammatory cells by optical microscopy. For each animal experimentation. All procedures described here had prior lung 10 fields (400×) were analyzed per section, and the data used to approval from the local animal ethics committee. calculate the mean number of cells per mm2. Mucus production was Fig. 2. Total cell counts and leukocyte quantification in BAL samples obtained from mice submitted to different treatments. Mice were sacrificed 24 h after the last challenge with ovalbumin. The cellularity in BAL fluid from control or ovalbumin-challenged mice treated with saline, dexamethasone (dexa) or umbelliferone (umb) was evaluated.